Mutant B-type DNA polymerases exhibiting improved performance in PCR

ABSTRACT

The present invention relates to thermostable mutants of B-type DNA polymerases comprising a Y-GG/A amino acid motif between the N-terminal 3′-5′-exonuclease domain and the C-terminal polymerase domain whereas the tyrosine of the Y-GG/A amino acid motif is mutated and whereas these mutant DNA polymerases are suitable for PCR.

[0001] The present application claims priority to co-pending EuropeanPatent Application No. 00105155.6, filed Mar. 11, 2000, which isincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] Subject of the invention is a thermostable mutant B-typeDNA-polymerase having a Y-GG/A amino acid motif between the N-terminal3′-5′-exonuclease domain and the C-terminal polymerase domain in thewild type form whereas amino acids of this motif are substituted in themutant form of the DNA polymerase and whereas these mutant DNApolymerases are suitable for PCR reactions. Thermostable mutantsaccording to the present invention exhibit better performance in PCRreactions compared to the wild type DNA polymerase. A further embodimentof the present invention is the use of these thermostable mutants of theB-type DNA polymerase for polymerase chain reactions (PCR) and othernucleic acid synthesizing reactions. Another subject of the presentinvention is a method of producing the inventive mutants, vectors andcell lines comprising genes encoding the inventive mutants.

BACKGROUND OF THE INVENTION

[0003] DNA-dependent DNA polymerases containing proof-reading activityhave to coordinate two catalytic activities: the DNA polymerase activityand the exonuclease activity. For polymerase I type DNA polymerases (E.coli Pol I) as well as for B-type DNA polymerases, these catalyticactivities are located on structurally distinct protein domains(Truniger, V., Lázaro, J., Salas, M. and Blanco, L. (1996) EMBO J.,15(13), 3430-3441; Pisani, F. M., De Felice, M. and Rossi, M. (1998)Biochemistry, 37(42), 15005-15012). In B-type (eukaryotic-type) DNApolymerases, the coordination of the two catalytic activities wasproposed to take place intramolecularly in the conserved motif Y-GG/Alocated between the N-terminal 3′-5′ exonuclease and the C-terminalpolymerization domain (Truniger, V., Lázaro, J., Salas, M. and Blanco,L. (1996) EMBO J., 15(13), 3430-3441; Pisani, F. M., De Felice, M. andRossi, M. (1998) Biochemistry, 37(42), 15005-15012). For the Klenowfragment of E.coli DNA polymerase it was described, that the editing canbe an intermolecular or intramolecular process involving dissociationand reassociation of the DNA depending on the local context (Joyce, C.M. (1989) JBC, 264(18), 10858-10866). Truniger et al. (Truniger, V.,Lázaro, J., Salas, M. and Blanco, L. (1996) EMBO J., 15(13), 3430-3441)demonstrated for the mesophile replicative DNA polymerase ofbacteriophage φ29 that mutations in the Y-GG/A motif can lead tophenotypes favoring either polymerisation or exonucleolysis compared tothe wild type enzyme. They could show that this effect is related toaltered (ss) DNA binding parameters and that the motif is important forthe communication between the polymerase and exonuclease active site ina combination of structural and functional roles.

[0004] For the DNA polymerase of the thermophilic crenarchaeonSulfolobus solfataricus (Sso) a region of 70 amino acids (region 1)involved in enzyme-DNA interaction was determined (Pisani, F. M., Manco,G., Carratore, V. and Rossi, M. (1996) Biochemistry, 35, 9158-9166). Itis located in the connecting part between the exonuclease domain and thepolymerase domain and contains the Y-GG/A motif. By mutational analysisof the amino acids in the Y-GG/A motif, it could be shown that the aminoacids in this part of the enzyme determine the processivity of theproofreading function (Pisani, F. M., De Felice, M. and Rossi, M. (1998)Biochemistry, 37(42), 15005-15012). Based on the crystal structure ofbacteriophage RB69 DNA polymerase, Truniger et al. proposed a directinteraction of the tyrosine with the phosphodiester bond between the twonucleotides preceding the one acting as template (Truniger, V., Blanco,L. and Salas, M. (1999) J. Mol. Biol., 286, 57-69).

DESCRIPTION OF THE INVENTION

[0005] The subject of the present invention was to provide thermostableDNA polymerases exhibiting an improved performance in PCR. Especially,thermostable mutants of a B-type DNA polymerase are provided whichexhibit improved PCR performance. The inventive mutants of the B-typeDNA polymerase have mutations in the Y-GG/A amino acid motif. Preferredmutations refer to the position of the tyrosine in the Y-GG/A amino acidmotif. Other mutations affecting the motif could also influence theperformance of B-type DNA polymerases in PCR.

[0006] According to the present invention an improved performance of aDNA polymerase in PCR is defined as a performance that results in higheryields of PCR product, or the amplification of longer DNA targets.Additionally, improved PCR performance can be defined as improvedfidelity during the amplification process.

[0007] Preferred mutant B-type DNA polymerases have mutations at theposition of the tyrosine in the Y-GG/A amino acid motif. Preferredmutants of B-type DNA polymerases according to the present inventionhave phenylalanine, tryptophan or histidine at the position of thetyrosine. Other preferred mutants of B-type DNA polymerases according tothe present invention have asparagine or serine at the position of thetyrosine. These mutant polymerases described here, in which the tyrosineof the Y-GG/A motif was substituted, exhibit an improved performance inPCR.

[0008] In a preferred embodiment the inventive mutant B-type DNApolymerase is a mutant of a B-type DNA polymerase obtainable fromEuryarchaea, more preferrably from Thermococcus aggregans (Tag).Especially preferred is a mutant of a B-type DNA polymerase from Tag ofabout 94 kDa size with a temperature optimum of ≧80° C. and the abilityto perform polymerase chain reactions.

[0009] The present invention is described in detail for the B-type DNApolymerase from Thermococcus aggregans, but the invention could also beapplied to other B-type DNA polymerases. Preferrably to those B-type DNApolymerases showing a high degree of homology (≧80%) to the DNApolymerase from Thermococcus aggregans.

[0010] The B-type DNA polymerase from Thermococcus aggregans exhibits ahigh degree of amino acid sequence homology to B-type DNA polymerases ofother Thermococcus species. The homology of the DNA polymerases wascalculated using the programm Blast 2 (Tatusova, T. A. and Madden, T. L.(1999) FEMS Microbiol. Lett. 174, 247-250). The homology of the B-typeDNA polymerase from Thermococcuis aggregans to the homologue enzymesfrom Thermococcus species is: 93% (T. litoralis), 87% (T. gorgonarius),86% (T. furiosus) and 87% (T. spec. 9N7). The homology of the Tag DNApolymerase to polymerases from Pyrococcus species is: 86% (P. abysii),86% (P. Horikoshii), 86% (P. spec KOD) and 85% (P. furiosus). A lowerhomology is calculated to other B-type DNA polymerase from differenteuryarchaeota: 59% (Methanococcus jannaschii), 56% (Methanococcusvoltae), 51% (Methanobacterium thermoautotrophicum) and 56%(Archaeglobus fulgidus). To B-type DNA polymerases from crenarchaeotaand bacteriophages the homology is found as follows: 46% (Sulfolobussolfataricus), 42% (Sulfolobus acidocaldarius), 41% (Sulfurispheraohwakuensis), 51% (Aeropyrum pernix), 40% (Pyrodictium occultum), 43%(Cenarchaeum symbiosum), 38% (bacteriophage T4) and 39% (bacteriophageRB69).

[0011] As described above several mutations in the Y-GG/A motif wereperformed for the Sulfolobus solfataricus (Sso) and the φ29 DNApolymerases. The observed effects on polymerase activity (pol) andexonuclease activity (exo) of these mutations do not completelycorrespond to the effects obtained for the Tag DNA polymerase. Thus theeffect of the mutations on the performance of the mutants in PCR was notpredictable.

[0012] The mutant Y387F of the Tag DNA polymerase exhibits a higherpol/exo ratio compared to the wild type Tag DNA polymerase. Similarresults were described for Sso and φ29 DNA polymerase. The mutant G389Adisplays the opposite effect than the corresponding mutant in φ29 DNApolymerase: while G→A in Tag DNA polymerase almost knocks out polymeraseactivity, in φ29 DNA polymerase G→A mutant this activity is clearlyenhanced. For mutants of the Sso DNA polymerase a change of exonucleaseprocessivity was described. Again, this was not observed for mutants ofthe B-type Tag DNA polymerases. Thus, a prediction of the effect ofanalogous mutants in the Y-GG/A motif could not be made.

[0013] In summary, although it has been described in the prior art thatthe Y-GG/A motif plays a role in the coordination of the DNA polymeraseactivity and the exonuclease activity, the observed changes of thepol/exo ratio of the prior art DNA polymerases do not strictly correlateto the changes observed for the inventive mutants of the Tag DNApolymerase. Furthermore, it has not been described that the Y-GG/A motifis important for the performance of B-type DNA polymerase in PCR.Additionally, there is no correlation between the changes of the pol/exoratio and the improvement of the performance of DNA polymerases in PCR.For instance, the mutant Y387H does not exhibit a change of pol/exoratio compared to the wild-type, but it exhibits improved performance inPCR. Furthermore, a significant enhancement of fidelity was observed forthe mutants Y387N and Y387S of Tag DNA polymerase.

[0014] Results obtained for the mutants of Tag DNA polymerase aredescribed in more detail below.

[0015] Enzymatic Activities of Wild Type and Mutant Tag DNA Polymerases

[0016] The enzymatic activities of the wild type enzyme and the mutantsof Tag DNA polymerase were determined and analyzed (FIG. 1). The DNApolymerase activity was determined as described in Example 2. Accordingto the effect of the mutations on the polymerase activity three groupsof mutants were defined: i) mutants with enhanced DNA polymeraseactivity (mutant Y387F), ii) mutants having a similar or slighltyreduced DNA polymerase activity compared to the wild type (mutants Y387Wand Y387H) and iii) mutants with reduced DNA polymerase activity(mutants Y387N, Y387S, G389A).

[0017] The exonuclease activity was determined as described in Example3. According to the effect of the mutations on the exonuclease activitytwo groups of mutants were defined: i) mutants with similarexonucleolytic activity as the wild type enzyme (mutants Y387F, Y387W,Y387H), ii) mutants with enhanced exonuclease activity (mutants Y387N,Y387S, G389A) in comparison to the wild type enzyme.

[0018] From the data obtained for polymerase activity and exonucleaseactivity the ratios of both activities (pol/exo) were calculated for thewild type enzyme and the mutants of Tag DNA polymerase (FIG. 1). Threemutants showed a higher or similar pol/exo ratio as the wild type enzyme(mutants Y387F, Y387W, Y387H). Three mutants showed a clearly reducedpol/exo ratio in comparison to the wild type enzyme (mutants Y387N,Y387S, G389A).

[0019] PCR Performance

[0020] Wild type and mutant enzymes were submitted to polymerase chainreactions on lambda DNA in a buffer optimized for this purpose. Allmutants except for mutant G389A were able to perform PCR, but yieldeddifferent amounts of product with a constant amount of enzyme (1 pmol).With increasing length of the DNA target, differences in performance ofthe enzyme were shown (FIG. 2). With 1 pmol of the mutants Y387S, Y387Nand G389A no PCR product could be obtained for the amplification of a3.3 kb fragment. 1 pmol of the wild type DNA polymerase could notamplify fragments of 5.0 kb length. The mutants Y387W, Y387F and Y387Hwere able to amplify a fragment of 7.5 kb length. As control Taq DNApolymerase, Pwo DNA polymerase and Expand™ High Fidelity PCR System(Roche Molecular Biochemicals) were used.

[0021] The differences in PCR performance were also shown by theamplification of a 2 kb fragment applying different elongation times inthe PCR runs. Under these conditions, all enzymes tested except themutant G389A were able to amplify a 2 kb fragment at an elongation timeof 90 sec/cycle. The mutants Y387F, Y387W and Y387H were able to amplifythe fragment at a reduced elongation time of 40 sec/cycle. The mutantY387H was able to amplify the target in a elongation time of 30sec/cycle (FIG. 3).

[0022] Exonuclease Processivity

[0023] The exonuclease processivity of the enzymes was studied in anexperiment based on the heparin trap method (Reddy, M. K., Weitzel, S.E. and von Hippel, P. H. (1992) J. Biol. Chem., 267(20), 14157-14166;Pisani, F. M., De Felice, M. and Rossi, M. (1998) Biochemistry, 37(42),15005-15012). A constant amount (1 pmol) of Tag DNA polymerase or itsmutants was incubated for 4 minutes at 68° C. with a 5′-DIG-labelled24mer oligonucleotide in the absence of nucleotides. In the absence ofheparin, the oligonucleotide was continually degraded by the Tag enzymes(positive control, FIG. 4, lanes “-”). The function of the heparin trapmethod was demonstrated by addition of heparin and MnCl₂ before thebinding of the enzyme (negative control, FIG. 4, lanes B). Singleturnover conditions (addition of heparin and MnCl₂ to start the reactionafter the binding of enzyme) resulted in exonucleolytic degradation ofthe oligonucleotide by the Tag DNA polymerases (FIG. 4, lanes A). Theenzymes showed differences in the exonucleolytic activity as shown bythe different amounts of remaining oligonucleotide that was notdegraded. However, for all enzymes tested the oligonucleotide wasdegraded to a similar extent (8 nt). This indicates a similarexonuclease processivity for the enzymes.

[0024] The Thermococcus gorgonarius DNA polymerase, which exhibits astrong exonuclease activity, was used as a positive control. It degradedthe 24mer oligonucleotide in the absence of heparin to oligonucleotidesof less than 15 bases length (FIG. 4, lane “-”). Under single turnoverconditions a strong degradation (11 nt) of the oligonucleotide isobserved (FIG. 4, lane “A”).

[0025] Fidelity

[0026] The error rates in amplification were determined for the mutantenzymes and the wild type DNA polymerase. The PCR-based fidelity assaydescribed by Frey and Suppman (Frey, M. and Suppmann, B. (1995)Biochemica, 2, 34-35) was used. This method is based on theamplification, circulation and transformation of the pUC19 derivativepUCQ17, which contains a functional lacL^(q) allele (Barnes, W. M.(1994) Proc. Natl. Acad. Sci. USA, 91, 2216-2220). PCR-derived mutationsin lad result in a de-repression of the expression of lacZα andsubsequent formation of a functional β-galactosidase enzyme, which canbe detected on X-Gal indicator plates.

[0027] In five independent runs, a mean error rate of 5.0×10⁻⁶ was foundfor the wild type Tag DNA polymerase. This value is in between the meanerror rates of 1.8×10 ⁻⁶ for Expand™High Fidelity PCR System (RocheMolecular Biochemicals) and 1.3×10⁻⁵ for Taq DNA polymerase (RocheMolecular Biochemicals) determined in the corresponding experiments. Fora better comparison of the data, we plotted the quotient of the errorrate determined for Taq DNA polymerase divided by the error ratesdetermined for the Tag

[0028] DNA polymerase and its mutants. In the independent experimentsthe error rate for Taq DNA polymerase varied from 1.2 to 3.05×10⁻⁵.

[0029]FIG. 5 shows the quotients of the error rates of the wild typeenzyme and mutants of Tag DNA polymerase. The error rates of the mutantsshowing improved PCR performance (Y387W, Y387F, Y387H) did notsignificantly differ from the values obtained for the wild type enzyme.The mutants with enhanced exonuclease activity (Y387N, Y387S) showedimproved fidelity rates (FIG. 5). For the mutants Y387N and Y387S meanerror rates of 6.3×10⁻⁷ and 6.2×10⁻⁷ were determined.

[0030] In contrast to the φ29 DNA polymerase and the Sso DNA polymerase,the Tag DNA polymerase, is suited for PCR. The mutant enzymes (Y387F,Y387W, Y387H) with an aromatic amino acid in the position of thetyrosine showed a similar or only slightly enhanced DNA polymeraseactivity (mutants Y387F, Y387W, Y387H) but an improvement in PCRperformance.

[0031] In the fidelity assay it was found that the mutants Y387F; Y387Wand Y387H showed no significant change in their error rate. By contrast,the mutants Y387N or Y387S showed higher exonuclease activity anddisplayed an improved fidelity.

[0032] Subject of the present invention is also a method of producingthe inventive B-type mutants comprising the following steps: cloning andmutagenesis of the gene, followed by the expression and purification ofthe protein.

[0033] Subject of the present invention is a DNA encoding for athermostable B-type DNA polymerase having a Y-GG/A amino acid motifbetween the N-terminal 3′-5′ exonuclease domain and the C-terminalpolymerase domain in the wild type enzyme whereas the tyrosine of thismotif is substituted in the mutant enzyme of the polymerase and whereasthis mutant DNA polymerase is suitable for PCR.

[0034] Preferably, said DNA in wild type form is obtainable fromEuryarchaea, more preferably from Thermococcus aggregans (Tag). Subjectof the present invention is also a vector containing the inventive DNA.Suitable vectors are e.g. the following: pET14b/15b/16b/19b (Novagen);pRSET (Invitrogen); pTrcHis (Invitrogen); pHAT10/11/12 (Clontech); pPROTet.E/Lar.A (Clontech); pCALn/n-EK (Stratagene); pGEMEX-1/-2 (Promega).

[0035] Furthermore, subjects of the present invention are also cellscomprising the above vector. Suitable cells are e.g. E.coli BL21,BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH5∝PRO, JM109 (DE3), TOP10in combination with the vectors recommended by the suppliers. The genemay have to be subcloned and the protein purification procedure may haveto be adapted in the case of different expression vectors.

[0036] A sample of the recombinant strain expressing Tag DNA polymerasewas deposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen (DSMZ) Mascheroder Weg 1b, D-38124 Braunschweig (DSM No.13224).

[0037] A further subject of the invention is the use of the inventivemutant enzymes for synthesizing nucleic acids e.g. in PCR reactions.

DETAILED DESCRIPTION OF THE PREFERED EMBODIMENTS

[0038] Figures:

[0039]FIG. 1.

[0040] Table showing the relative polymerase activities (Pol) and3′-5′-exonuclease activities (Exo) of Tag DNA polymerase and its mutantson double-stranded DNA.

[0041] Assays were carried out as described in example 2 and 3,respectiviely. The activites are expressed as percentage of the activityobtained for the wild-type Tag DNA polymerase.

[0042]FIG. 2.

[0043] PCR With Tag DNA Polymerase Mutants.

[0044] Tag DNA polymerase mutants (1 pmol) were incubated in a 50 μltotal volume with 10 ng of lambda DNA as template and 30 pmoles of aprimer set designed to yield the indicated fragment lengths, 200 μMdNTPs and the suitable PCR buffer. Reactions were performed with 10cycles of 10 sec 94° C., 30 sec 57° C. and 3.0 min (A), 4.3 min (B) or7.0 (C) min of elongation time at 72° C. followed by 20 cycles withelongation times increasing by 20 sec/cycle. After the PCR 5 μl samplewere submitted to electrophoresis on a 1% agarose gel. For the controlreaction 2.5 U of Taq DNA polymerase, Pwo DNA polymerase or Expand™ HighFidelity PCR System were used. The labeling of the lanes is described inthe legend of FIG. 6.

[0045]FIG. 3.

[0046] Time-dependent Polymerase Chain Reaction.

[0047] 1% agarose gels showing 2 kb PCR products from reactionsperformed with different elongation time (90 sec, 40 sec, 30 sec asindicated) to determine the minimal elongation time. 1 pmol of each TagDNA polymerase mutant or wild type enzyme was added to a mix of 10 nglambda DNA and primers designed to yield 2 kb DNA fragments. Labeling ofthe lanes is described in the legend of FIG. 6. For each mutantduplicate reactions were performed. Right lane of each gel Pwo: 2.5 UPyrococcus woesei DNA polymerase (Roche Molecular Biochemicals) ascontrol reaction. Left lane of each gel: Molecular weight marker VI(Roche Molecular Biochemicals). In the 40 sec reaction, mutant GA wasomitted. In the 30 sec reaction, for the mutant YS only one reaction wasrun on the gel.

[0048]FIG. 4.

[0049] 3′-5′-exonuclease Processivity.

[0050] The Tag DNA polymerase mutants tested are indicated on top of thefigure. Tgo DNA polymerase was used as a control reaction (incubationfor 30 sec). Reactions for wild type and mutants of Tag DNA polymerasewere performed for 4 minutes at 68° C. after preincubation for 1 minuteat 68° C. Lane “P” is the control reaction (24 mer 5′-DIG-labelledprimer without incubation), lane “-”: reaction without heparin (positivecontrol); lane “B”: reaction with heparin and MnCl₂ added beforeaddition of the enzyme (negative control); lane “A”: heparin and MnCl₂added after the enzyme (reaction under single turnover conditions).

[0051]FIG. 5.

[0052] Fidelity of Tag DNA Polymerase Mutants.

[0053] The fidelity of Tag DNA polymerase and its mutants was expressedin relation to the fidelity of Taq DNA polymerase. A quotient of 1 meansthat the polymerase has the same error rate as Taq DNA polymerase (meanvalue 1.3×10⁻⁵). Values >1 reflect the factor by which a polymeraseshows less errors than Taq DNA polymerase. The bars correspond to tomean values calculated from 2-5 independent experiments, error barsmissing are smaller than 0.36. Abbreviations for enzymes are asindicated in legend to FIG. 6. As controls Pyrococcus woesei DNApolymerase (Roche Molecular Biochemicals) and Expand High Fidelity PCRSystem (Roche Molecular Biochemicals) were used (,,Taq/Pwo” and,,Taq/HiFi”).

[0054]FIG. 6.

[0055] SDS-PAGE Gel Analysis of Purified Mutant Proteins.

[0056] 1 μg of each mutant was submitted to electrophoresis on an 10%SDS-PAGE gel. Left: MW, molecular weight marker; WT, Thermococcusaggregans wild type DNA polymerase; YF, YW, YS, YN, YH are thecorresponding mutants with an exchange at the position of tyrosine 387to phenylalanine, tryptophan, serine, asparagine, histidine,respectively. GA, mutation of glycine 389 to alanine in the gene of theThermococcus aggregans DNA polymerase. All mutants showed the samechromatographic behaviour and solubility as the wild type enzyme.

[0057]FIG. 7.

[0058] Qualitative Exonuclease Assay.

[0059] A DNA molecular weight marker was used as substrate to test theexonucleolytic activity (DNA molecular weight marker II (MW II), RocheMolecular Biochemicals). 1 μg of MW II was incubated for 6 h at 65° C.with 1 pmol of each variant of Tag DNA polymerase in the presence (A) orabsence (B) of 200 μM dNTP. Tag mutants are named as explained in legendof FIG. 6. Exonucleolytic degradation take place only in the absence ofdeoxynucleotides. The qualitative ranking of the proteins in terms ofexonuclease activity is GA>YN>YS>YH>YF=YW=WT.

[0060]FIG. 8.

[0061] Consensus sequence motif for B-type DNA polymerases from theorder of Thermococcales derived from a multiple alignment of amino acidsequences of euryarchaeal and crenarchaeal B-type DNA polymerases.

[0062] A region of 24 amino acids containing the Y-GG/A motif wasanalyzed with the ClustalW Software program (Higgins, EMBL Heidelberg,Germany). In addition to the amino acids conserved in all archaealB-type DNA polymerases (like the Y-GG/A motif), a consensus sequence“E--RR-R-----G(Y)-KE-EE--LWE-” can be defined. This sequence is found inthe sequence of all DNA polymerases belonging to the order of theThermococcales and coincides with a homology of >80% of the DNApolymerases.

[0063] The sequences of the crenarchaeal species Sulfolobussolfataricus, Sulfolobus acidocaldarius, Pyrobaculum islandicum,Pyrodictium occultum, Aeropyrum pernix, Sulfurisphaera ohwakuensis andthe sequences of several euryarchaeal species Thermococcus (“T.”),Pyrococcus (“P.”) and Methanococcus (“M.”) were aligned.

[0064]FIG. 9.

[0065] DNA sequence and deduced amino acid sequence of recombinant wildtype Tag DNA polymerase.

[0066] Three inteins found in the native gene (Acc. No. Y13030) weredeleted by PCR (Niehaus, F., Frey, B., Antranikian, A. (1997) Gene, 204,153-158). Four mutations leading to amino acid exchanges were introducedduring PCR. The amino acid exchanges (native→recombinant) are: L3F,A404T, S410C and L492H.

EXAMPLE 1

[0067] Site-directed Mutagenesis and Expression of Tag DNA PolymeraseMutants

[0068] The cloning of the gene of Tag DNA Polymerase (polTY) wasdescribed earlier (Niehaus, F., Frey, B., Antranikian, A. (1997) Gene,204, 153-158). Overexpression of Tag DNA Polymerase in E. coli wasachieved by subcloning its encoding gene into the IPTG-inducible pET15bvector (Novagen) containing an N-terminal His-Tag for purification (theresulting plasmid was named pET15b-TagPol).

[0069] The mutants presented in this study were prepared in polymerasechain reactions using primers containing the desired mutations as amismatch. The forward primer was universally “Kpn-fw”, matching to asequence about 100 bp upstream of the mutation site and contained a KpnIrestriction site of the polTy gene. The reverse primers contained aSnaBI restrition site and additionally the desired mutation. Thesequences of the oligonucleotides were as follows (mismatch sites formutagenesis underlined):

[0070] SEQ. ID. NO: 1

[0071] Kpn-Fw 5′-GCAACCTTGTAGAGTAGAGTGGTACCTGTTAAGGG-3′;

[0072] SEQ. ID. NO: 2

[0073] TagY387F 5′-GCCTCTTTCCGGCTCTTTTACGTATCCTCCCAGGAAAGTAGTCC-3′,

[0074] SEQ. ID. NO:3

[0075] TagY387H 5′-GCCTCTTTCCGGCTCTTTTACGTATCCTCCCAGGTGAGTAGTCC-3′,

[0076] SEQ. ID. NO: 4

[0077] TagY387N 5′-GCCTCTTTCCGGCTCTTTTACGTATCCTCCCAGGTTAGTAGTCC-3′,

[0078] SEQ. ID. NO: 5

[0079] TagY387S 5′-GCCTCTTTCCGGCTCTTTTACGTATCCTCCCAGGGAAGTAGTCC-3′,

[0080] SEQ. ID. NO: 6

[0081] TagY387W 5′-GCCTCTTTCCGGCTCTTTTACGTATCCTCCCAGCCAAGTAGTCC-3′,

[0082] SEQ. ID. NO: 7

[0083] TagG389A 5′-GCCTCTTTCCGGCTCTTTTACGTATCCAGCCAGGTAAGTAGTCC-3′.

[0084] PCR reactions were carried out with Expand™ High Fidelity PCRSystem (Roche Molecular Biochemicals) using the following program: 2 min94° C., 30 cycles of 10 sec 94° C., 30 sec 55° C., 30 sec at 72° C. Theresulting 139 bp fragments were digested with the restriction enzymesKpnI and SnaBI yielding a 101 bp fragment that was ligated intopET15b-TagPol linearized with the restriction enzymes KpnI and SnaBI.The cloned DNA fragments were sequenced to confirm the presence of thedesired mutations.

[0085] For protein expression, E. coli BL21 (DE3) cells were transformedwith the expression vector pET15b-TagPol. Three to five colonies wereinoculated in 15 ml of LB medium supplemented with 100 μg Ampicillin perml and grown to OD_(600nm) 0.3. An aliquot (10 ml) of the preculture wasused to inoculate 500 ml of LB medium and incubated while shaking at 37°C. At OD_(600nm=)0.6 expression was induced by addition of IPTG (finalconcentration: 1 mM). After incubation for 3 hours cells were harvestedby centrifugation and suspended in 50 mM Tris-HCl/pH 7.5, 10 mM KCl, 0.5mM EDTA, 4 mM MgCl₂, 5 mM DTT. Cells were sonicated on ice and the crudeextract was heated for 15 min to 80° C. Cell debris was removed bycentrifugation (30 min, 30000× g at 4° C.).

[0086] The supernatant was applied to a Blue Sepharose 3G-A column(Pharmacia) equilibrated with buffer A (50 mM Tris-HCl/pH 7.5, 10 mMKCl, 4 mM Mg Cl₂). The protein was eluted with a gradient of 0.01-1.5 MKCl. Active fractions were pooled and dialyzed against 20 mM Tris-HCl/pH7.9, 5 mM imidazole, 500 mM NaCl. The sample was applied to a Ni-chelatecolumn (Novagene) equilibrated in the same buffer and eluted with agradient of 0.005-1 M imidazole. Active fractions were pooled anddialyzed against storage buffer (50 mM Tris-HCl/pH 7.5, 100 mM KCl, 0.5mM EDTA, 5 mM DTT, 50% glycerol). The enzymes were pure as shown by SDSgel electrophoresis (FIG. 6).

EXAMPLE 2

[0087] DNA Polymerase Assay

[0088] The DNA polymerase activity was determined by measuring theincorporation of α-(³²P)dCTP in a DNA substrate. The test mix (50 μl)contained 5 μl 10× Tag reaction buffer (100 mM Tris-HCl/pH 8.9, 750 mMKCl, 15 MgCl₂, 100 mM CHAPS), 200 μM of each dATP, dGTP, dTTP, 100 μMdCTP, 1 mCi α-(³²P)dCTP, 1 μg of M13mp9 ssDNA annealed with 0.3 μg M13primer. Assays were performed with 2 and 3 μl of enzyme in threedifferent dilutions (final amount of enzyme 2.5 to 15 fmoles) yieldingsix reactions to calculate a mean value. As a reference Pwo DNApolymerase was used. The DNA/primer mix was prepared by heating 277.2 μgM 13mp9 ssDNA (Roche Molecular Biochemicals) and 156 μg M13 sequencingprimer (17mer forward primer, Roche Molecular Biochemicals) for 30minutes to 55° C. and then cooling it for 30 minutes to roomtemperature.

[0089] Assay reactions were incubated for 30 minutes at 65° C., stoppedon ice by addition of 500 PI of 10% TCA (4° C.) and kept on ice foranother 10 minutes. Samples were filtered over GFC-filter (Whatman),filters washed three times with 5% TCA, dried and submitted toβ-counting in 2 ml of scintillation fluid. One unit is defined as theamount of enzyme necessary to incorporate 10 nM dNTP into acid insolublematerial at 65° C. in 30 minutes.

EXAMPLE 3

[0090] Exonuclease Assays

[0091] Activity Assay

[0092] 3 μl (300 ng) of enzyme (approximately 5 Units of polymeraseactivity) were incubated with 5 μg of 3H-labelled calf thymus DNA for 4hours at 65° C. in a buffer containing 10 mM Tris-HCl/pH 8.9, 75 mM KCl,1.5 MgCl₂, 10 mM CHAPS. Radioactivity liberated from calf thymus DNA wasmeasured in a scintilation counter.

[0093] The assay used does not discriminate between the 3′-5′exonuclease activity and the 5′-3′ exonuclease activity.5′-3′-exonuclease activity has not been detected pheno- or genotypicallyin B-type polymerases of Thermococcales (Perler, F. B., Kumar, S. andKong, H. (1996) Adv. Prot. Chem., 48, 377-435). Thus the values obtainedcan be regarded as 3′-5′-exonuclease activity.

[0094] In another assay, 1 μg of molecular weight marker II (RocheMolecular Biochemicals) were incubated in same buffer as above with 5 Uof the indicated protein with or without 200 μM dNTP in a final volumeof 50 μl for 6 hours at 65° C. The reaction products were separated byelectrophoresis on 1% agarose gels.

[0095] 3′-5′ Exonuclease Processivity Assay

[0096] The previously described heparin trap method was used (Reddy, M.K., Weitzel, S. E. and von Hippel, P. H. (1992) J. Biol. Chem., 267(20),14157-14166; Pisani, F. M., De Felice, M. and Rossi, M. (1998)Biochemistry, 37(42), 15005-15012). A reaction mix (10 μl) containing 10mM Tris-HCl/pH 8.9, 75 mM KCl, 10 mM CHAPS and 0.5 pmoles of a5μ-DIG-labeled 24mer oligonucleotide was prewarmed for 1 minute at 68°C. in a thermocycler. Unless otherwise noted, 1 pmol of the enzyme waspreincubated for 1 minute at 68° C. with the substrate. The reaction wasstarted by addition of MnCl₂ (final concentration: 4 mM) and heparin(final concentration:1 mg/ml) to ensure single turnover conditions.After incubation for 4 minutes, the reaction was stopped by the additionof 5 μl of formamide buffer (80% formamide, 10 mM EDTA, 1 mg/mlbromophenol blue, 1 mg/ml xylene xyanol). The efficiency of the heparintrap was checked in a control reaction by adding heparin and MnCl₂ priorto the addition of the enzyme. The samples were denaturated for 3minutes at 90° C. and subjected to denaturing gel electrophoresis on a17.5% polyacrylamide/8 M urea gel. Gels were blotted to a positivelycharged nylon membrane (Roche Molecular Biochemicals) and the blotsdeveloped with CPD-Star (Roche Molecular Biochemicals) according to themanufacturers instructions.

EXAMPLE 4

[0097] lacI-based PCR Fidelity Assay

[0098] We used the lad-based PCR fidelity assay described by Frey andSuppmann (Frey, M. and Suppmann, B. (1995) Biochemica, 2, 34-35). Thismethod is based on the amplification, circularization and transformationof the pUC19 derivative pUCQ17, which contains a functional lacI^(q)allele (Barnes, W. M. (1994) Proc. Natl. Acad. Sci. USA, 91, 2216-2220).PCR-derived mutations in lacI result in a de-repression of theexpression of lacZα and subsequent formation of a functionalβ-galactosidase enzyme, which can be easily detected on X-Gal indicatorplates.

[0099] The truncated lacI gene of pUC19 was substituted by a functionalcopy of lacI^(q). A 178 bp Pvu II-Afl III fragment was replaced by a1121 bp DNA fragment encoding lacI^(q). The α-complementing E. colistrain DH5α, once transformed with the resulting plasmid pUCIQ17 (3632bp), produces white (LACI⁺) colonies on LB plates containing ampicillin(100 μg/ml) and X-Gal (0.004% w/v). For the PCR, pUCIQ17 was linearizedby digestion with Dra II and used as a template in an amount of 1 or 10ng. Both primers have Cla I cleavage sites at their 5′ ends.Oligonucleotide Cla33 (34mer, 24 matches: SEQ. ID. NO: 8: 5′-AGC TTA TCGATG GCA CTT TTC GGG GAA ATG TGC G-3′) and Oligonucleotide Cla55 (36mer,26 matches: SEQ. ID. NO: 9: 5′-AGC TTA TCG ATA AGC GGA TGC CGG GAG CAGACA AGC-3′) resulted in a PCR product of 3493 bp. The reactions wereperformed with 1 or 5 pmol of protein in the Tag polymerase PCR bufferdescribed below or for the control reactions in the manufacturers PCRbuffers with 2.5 U of enzyme. The cycle conditions were 10 secdenaturation at 94° C., 30 sec annealing at 57° C. and 4 min elongationat 72° C. for 18, 24 or 30 cycles depending on the enzyme.

[0100] After PCR, the yield of amplification product was determined at(OD_(260nm) or in agarose gel) and the DNA fragments submitted tophenol/chloroform extraction to eliminate any protein. After digestionwith ClaI, the DNA fragments were purified from a preparative agarosegel. Ligation reactions were carried out with the Rapid Ligation Kit(Roche Molecular Biochemicals), the reactions contained 30 ng DNA. Theresulting circular plasmids were transformed in E. coli DH5α asdescribed by Hanahan (Hanahan, D. (1983) J. Mol. Biol., 166, 557-580)and plated on LB Amp/X-Gal plates described above. After incubationovernight at 37° C., blue and white colonies were counted. The errorrate (f) per bp was calculated with a rearranged equation published byKeohavong and Thilly (Keohavong, P. and Thilly, W. G. (1989) Proc. Natl.Acad. Sci. USA, 86, 9253-9257): f=−lnF/d×b bp.

[0101] Where F is the fraction of white colonies (white colonies/totalcolonies); d is the number of DNA duplications: 2^(d)=output DNA/inputDNA and b is the effective target size (1080 bp) of the lad gene. Thereare 349 phenotypically identified (by colour screening) single-basesubstitutions (non-sense and mis-sense) at 179 codons (approximately 50%of the coding region) within the lad gene. (Provost, G. S., Kretz, P.L., Hamner, R. T., Matthews, C. D., Rogers, B. J., Lundberg, K. S.,Dycaico, M. J. and Short, J. M. (1993) Mut. research, 288, 133-149).Frameshift errors which may occur at every position in the 1080 bp openreading frame of lad, are not taken into account because littleinformation is available for the specific polymerases used in PCRsystems except for Taq DNA polymerase.

EXAMPLE 5

[0102] Polymerase Chain Reactions

[0103] PCR was performed in a buffer optimized for Tag DNA polymeraseand its mutants: 10 mM Tris-HCl/pH 8.9, 75 mM KCl, 1.5 MgCl₂, 10 mMCHAPS, 200 μM dNTP. 10 ng of λ DNA were used as a template and 30 pmolof each primer (20 bp, designed to yield the products of the desiredlength): Lambda 1, universal 5′-GAT GAG TTC GTG TCC GTA CAA CA-3′, SEQ.ID. NO: 10, forward primer: Lambda 3.3: 5′-CTC ATC AGC AGA TCA TCT TCAGG-3′, SEQ. ID. NO: 11, Lambda 8: 5′-ACT CCA GCG TCT CAT CTT TAT GC-3′,SEQ. ID. NO: 12, Lambda 9: 5′-GAT GGT GAT CCT CTC TCG TTT GC-3′, SEQ.ID. NO: 13.

[0104] Lambda 3.3, 8 and 9 were used as reverse primers for theamplification of 3.3 kb, 5 kb and 7.5 kb fragments respectively.

[0105] Template, primers and nucleotides were prepared in mix 1 in avolume of 25 μl. Then 25 μl of mix 2 containing the buffer and enzyme (1pmol Tag wild type or mutant; or 2.5 U control enzyme) were added. Allreactions were prepared in duplicate. The amplification was performed ina 2400 GeneAmp thermocycler (Perkin Elmer). The cycle conditions were: 2min at 94° C., 10 cycles with 10 sec denaturation at 94° C., 30 secannealing at 58° C. and elongation at 72° C.

[0106] Elongation times depended on the length of the product (3 min for3.3 kb, 4.3 min for 5 kb and 7 min for 7.5 kb). Another 20 cycles wereperformed with increasing the elongation times by 20 sec/cycle. Thereaction was finished by 7 minutes at 72° C. The tubes were kept at 4°C. until separation by electrophoresis on 1% agarose gels.

[0107] The improvement of the PCR performance of the mutants was alsostudied in a “time-dependent PCR”. A 2 kb fragment was amplified fromlambda DNA as described above. In these studies the elongation time ofthe PCR was stepwise reduced (90 sec, 40 sec, 30 sec) to determine foreach enzyme the minimal elongation time that was sufficient to amplifythe 2 kb fragment. The following primers were used: Lambda 1, universal5′-GAT GAG TTC GTG TCC GTA CAA CA-3′, SEQ. ID. NO: 14, forward primer:Lambda 6, reverse 5′-CTT CAT CAT CGA GAT AGC TGT CG-3′, SEQ. ID. NO: 15.primer:

[0108] The temperature profile was as described above. The elongationtimes were kept constant over 30 cycles.

1 34 1 35 DNA Artificial amplification primer 1 gcaaccttgt agagtagagtggtacctgtt aaggg 35 2 44 DNA Artificial amplification primer 2gcctctttcc ggctctttta cgtatcctcc caggaaagta gtcc 44 3 44 DNA Artificialamplification primer 3 gcctctttcc ggctctttta cgtatcctcc caggtgagta gtcc44 4 44 DNA Artificial amplification primer 4 gcctctttcc ggctcttttacgtatcctcc caggttagta gtcc 44 5 44 DNA Artificial amplification primer 5gcctctttcc ggctctttta cgtatcctcc cagggaagta gtcc 44 6 44 DNA Artificialamplification primer 6 gcctctttcc ggctctttta cgtatcctcc cagccaagta gtcc44 7 44 DNA Artificial amplification primer 7 gcctctttcc ggctcttttacgtatccagc caggtaagta gtcc 44 8 34 DNA Artificial amplification primer 8agcttatcga tggcactttt cggggaaatg tgcg 34 9 36 DNA Artificialamplification primer 9 agcttatcga taagcggatg ccgggagcag acaagc 36 10 23DNA Artificial amplification primer 10 gatgagttcg tgtccgtaca aca 23 1123 DNA Artificial amplification primer 11 ctcatcagca gatcatcttc agg 2312 23 DNA Artificial amplification primer 12 actccagcgt ctcatcttta tgc23 13 23 DNA Artificial amplification primer 13 gatggtgatc ctctctcgtttgc 23 14 23 DNA Artificial amplification primer 14 gatgagttcgtgtccgtaca aca 23 15 23 DNA Artificial amplification primer 15cttcatcatc gagatagctg tcg 23 16 25 PRT T. aggregans 16 Glu Tyr Arg ArgArg Leu Arg Thr Thr Tyr Leu Gly Gly Tyr Val Lys 1 5 10 15 Glu Pro GluArg Gly Leu Trp Glu Asn 20 25 17 25 PRT T. litoralis 17 Glu Tyr Lys ArgArg Leu Arg Thr Thr Tyr Leu Gly Gly Tyr Val Lys 1 5 10 15 Glu Pro GluLys Gly Leu Trp Glu Asn 20 25 18 24 PRT T. fumicolans 18 Glu Leu Glu ArgArg Arg Gly Gly Tyr Ala Gly Gly Tyr Val Lys Glu 1 5 10 15 Pro Glu ArgGly Leu Trp Glu Asn 20 19 24 PRT T. spec. 9N7 19 Glu Leu Ala Arg Arg ArgGly Gly Tyr Ala Gly Gly Tyr Val Lys Glu 1 5 10 15 Arg Glu Arg Gly LeuTrp Glu Asn 20 20 24 PRT T. gorgonarius 20 Glu Leu Ala Arg Arg Arg GluSer Tyr Ala Gly Gly Tyr Val Lys Glu 1 5 10 15 Pro Glu Arg Gly Leu TrpGlu Asn 20 21 24 PRT P. spec. KOD 21 Glu Leu Ala Arg Arg Arg Gln Ser TyrGlu Gly Gly Tyr Val Lys Glu 1 5 10 15 Pro Glu Arg Gly Leu Trp Glu Asn 2022 25 PRT P. abysii 22 Glu Tyr Glu Arg Arg Leu Arg Glu Ser Tyr Glu GlyGly Tyr Val Lys 1 5 10 15 Glu Pro Glu Lys Gly Leu Trp Glu Asn 20 25 2325 PRT P. furiosus 23 Glu Tyr Gln Arg Arg Leu Arg Glu Ser Tyr Thr GlyGly Phe Val Lys 1 5 10 15 Glu Pro Glu Lys Gly Leu Trp Glu Asn 20 25 2425 PRT P. horikoshii 24 Glu Tyr Glu Arg Arg Leu Arg Glu Ser Tyr Glu GlyGly Tyr Val Lys 1 5 10 15 Glu Pro Glu Lys Gly Leu Trp Glu Asn 20 25 2525 PRT M. jannaschii 25 Glu Tyr Arg Arg Arg Val Leu Thr Thr Tyr Glu GlyGly Tyr Val Lys 1 5 10 15 Glu Pro Glu Lys Gly Met Phe Glu Asp 20 25 2625 PRT M. voltae 26 Ser Tyr Arg Glu Arg Ala Lys Phe Ser Tyr Glu Gly GlyTyr Val Arg 1 5 10 15 Glu Pro Leu Lys Gly Ile Gln Glu Asn 20 25 27 25PRT S. solfataricus 27 Thr Ser Ala Leu Ile Lys Gly Lys Gly Tyr Lys GlyAla Val Val Ile 1 5 10 15 Asp Pro Pro Ala Gly Ile Phe Phe Asn 20 25 2825 PRT S. acidocaldarius 28 Thr Ala Ala Val Ile Lys Gly Lys Lys Tyr LysGly Ala Val Val Ile 1 5 10 15 Asp Pro Pro Ala Gly Val Tyr Phe Asn 20 2529 25 PRT P. islandicum 29 Thr Lys Ala Ile Ile Lys Gly Lys Lys Tyr AlaGly Ala Val Val Leu 1 5 10 15 Asp Pro Pro Leu Gly Ile Phe Phe Asn 20 2530 25 PRT P. occultum 30 Ser Glu Ala Leu Ile Lys Gly Lys Lys Tyr Gln GlyAla Leu Val Leu 1 5 10 15 Asp Pro Pro Ser Gly Ile Tyr Phe Asn 20 25 3125 PRT A. pernix 31 Val Gly Ala Ile Ile Lys Asp Lys Lys Tyr Arg Gly AlaIle Val Leu 1 5 10 15 Asp Pro Pro Val Gly Ile Phe Phe Arg 20 25 32 25PRT S. chwakuensis 32 Thr Ala Ala Ile Ser Lys Gly Lys Arg Tyr Lys GlyAla Val Val Ile 1 5 10 15 Asp Pro Pro Ala Gly Val Phe Phe Asn 20 25 332325 DNA T. aggregans CDS (1)..(2325) 33 atg ata ttt gac act gac tac ataaca aag gac ggt aaa ccc ata att 48 Met Ile Phe Asp Thr Asp Tyr Ile ThrLys Asp Gly Lys Pro Ile Ile 1 5 10 15 cga att ttc aag aaa gag aac ggggaa ttt aaa ata gaa ctt gat cca 96 Arg Ile Phe Lys Lys Glu Asn Gly GluPhe Lys Ile Glu Leu Asp Pro 20 25 30 cat ttt cag ccc tac att tac gct cttctc aaa gat gac tcc gct att 144 His Phe Gln Pro Tyr Ile Tyr Ala Leu LeuLys Asp Asp Ser Ala Ile 35 40 45 gat gaa ata aaa gca ata aaa ggc gag agacac gga aaa att gtg aga 192 Asp Glu Ile Lys Ala Ile Lys Gly Glu Arg HisGly Lys Ile Val Arg 50 55 60 gta gtc gat gca gtg aaa gtc aag aag aaa tttttg ggg aga gat gtt 240 Val Val Asp Ala Val Lys Val Lys Lys Lys Phe LeuGly Arg Asp Val 65 70 75 80 gag gtc tgg aag ctt ata ttt gag cat ccc caagac gtc ccg gcc cta 288 Glu Val Trp Lys Leu Ile Phe Glu His Pro Gln AspVal Pro Ala Leu 85 90 95 agg ggc aag ata agg gaa cat cca gct gtg att gacatt tat gag tac 336 Arg Gly Lys Ile Arg Glu His Pro Ala Val Ile Asp IleTyr Glu Tyr 100 105 110 gac ata ccc ttt gcc aag cgc tac ctc ata gac aagggc ttg atc cct 384 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys GlyLeu Ile Pro 115 120 125 atg gag ggc gac gag gag ctt aag cta atg gcc ttcgac att gag acg 432 Met Glu Gly Asp Glu Glu Leu Lys Leu Met Ala Phe AspIle Glu Thr 130 135 140 ttt tac cac gag gga gac gag ttt ggg aag ggc gagata ata atg ata 480 Phe Tyr His Glu Gly Asp Glu Phe Gly Lys Gly Glu IleIle Met Ile 145 150 155 160 agc tac gcc gat gag gaa gag gca agg gta attaca tgg aag aat att 528 Ser Tyr Ala Asp Glu Glu Glu Ala Arg Val Ile ThrTrp Lys Asn Ile 165 170 175 gat ctg ccc tac gtt gat gtt gta tcc aac gaaagg gag atg ata aag 576 Asp Leu Pro Tyr Val Asp Val Val Ser Asn Glu ArgGlu Met Ile Lys 180 185 190 cgg ttt gtg caa att gtc agg gaa aaa gac ccggat gtc ctg ata act 624 Arg Phe Val Gln Ile Val Arg Glu Lys Asp Pro AspVal Leu Ile Thr 195 200 205 tac aat gga gac aac ttt gat ttg ccg tac cttata aaa agg gca gag 672 Tyr Asn Gly Asp Asn Phe Asp Leu Pro Tyr Leu IleLys Arg Ala Glu 210 215 220 aag tta gga gtt act ctt ctc ttg ggg agg gacaaa gaa cac ccc gag 720 Lys Leu Gly Val Thr Leu Leu Leu Gly Arg Asp LysGlu His Pro Glu 225 230 235 240 ccc aag att cac aga atg ggc gat agc tttgcc gtg gaa att aaa ggc 768 Pro Lys Ile His Arg Met Gly Asp Ser Phe AlaVal Glu Ile Lys Gly 245 250 255 aga att cac ttt gat ctc ttc ccg gtt gtgcgg aga acc ata aac ctc 816 Arg Ile His Phe Asp Leu Phe Pro Val Val ArgArg Thr Ile Asn Leu 260 265 270 cca aca tac acg ctt gag gca gtt tat gaagcc gtc ttg gga aaa acc 864 Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu AlaVal Leu Gly Lys Thr 275 280 285 aaa agc aag ctg ggt gcg gag gaa atc gccgcc atc tgg gaa aca gag 912 Lys Ser Lys Leu Gly Ala Glu Glu Ile Ala AlaIle Trp Glu Thr Glu 290 295 300 gag agc atg aag aag ctg gcc cag tac tcgatg gaa gat gct agg gca 960 Glu Ser Met Lys Lys Leu Ala Gln Tyr Ser MetGlu Asp Ala Arg Ala 305 310 315 320 act tat gaa ctc gga aaa gag ttt ttcccc atg gag gca gag cta gca 1008 Thr Tyr Glu Leu Gly Lys Glu Phe Phe ProMet Glu Ala Glu Leu Ala 325 330 335 aag cta ata ggc caa agc gta tgg gacgtc tca aga tca agc act ggc 1056 Lys Leu Ile Gly Gln Ser Val Trp Asp ValSer Arg Ser Ser Thr Gly 340 345 350 aac ctt gta gag tgg tac ctg tta agggtg gca tat gag agg aat gag 1104 Asn Leu Val Glu Trp Tyr Leu Leu Arg ValAla Tyr Glu Arg Asn Glu 355 360 365 ctc gct ccg aac aag ccg gat gaa gaagag tac aga agg cgt tta agg 1152 Leu Ala Pro Asn Lys Pro Asp Glu Glu GluTyr Arg Arg Arg Leu Arg 370 375 380 act act tac ctg gga gga tac gta aaagag ccg gaa aga ggc tta tgg 1200 Thr Thr Tyr Leu Gly Gly Tyr Val Lys GluPro Glu Arg Gly Leu Trp 385 390 395 400 gag aac atc acc tat tta gac tttagg tgc cta tac ccc tca att ata 1248 Glu Asn Ile Thr Tyr Leu Asp Phe ArgCys Leu Tyr Pro Ser Ile Ile 405 410 415 gtt acc cac aac gtc tcc cct gacact tta gaa aga gaa ggc tgc aag 1296 Val Thr His Asn Val Ser Pro Asp ThrLeu Glu Arg Glu Gly Cys Lys 420 425 430 aat tac gat gtt gcc ccg ata gtaggt tat aag ttc tgc aag gat ttt 1344 Asn Tyr Asp Val Ala Pro Ile Val GlyTyr Lys Phe Cys Lys Asp Phe 435 440 445 ccc ggt ttc att cca tct ata ctcggg gaa tta atc aca atg agg caa 1392 Pro Gly Phe Ile Pro Ser Ile Leu GlyGlu Leu Ile Thr Met Arg Gln 450 455 460 gaa ata aag aag aag atg aaa gctaca att gac cca ata gaa aag aaa 1440 Glu Ile Lys Lys Lys Met Lys Ala ThrIle Asp Pro Ile Glu Lys Lys 465 470 475 480 atg ctt gat tat agg caa agagct gtt aaa ttg cac gca aac agc tat 1488 Met Leu Asp Tyr Arg Gln Arg AlaVal Lys Leu His Ala Asn Ser Tyr 485 490 495 tac ggt tat atg ggc tat cccaag gcg agg tgg tac tcg aag gaa tgt 1536 Tyr Gly Tyr Met Gly Tyr Pro LysAla Arg Trp Tyr Ser Lys Glu Cys 500 505 510 gcc gaa agc gtt acc gcg tgggga agg cac tac ata gaa atg acc ata 1584 Ala Glu Ser Val Thr Ala Trp GlyArg His Tyr Ile Glu Met Thr Ile 515 520 525 aaa gag ata gag gag aaa tttgga ttt aag gtg cta tat gcc gac act 1632 Lys Glu Ile Glu Glu Lys Phe GlyPhe Lys Val Leu Tyr Ala Asp Thr 530 535 540 gat ggt ttt tac gcc aca ataccg gga gaa aaa cct gaa aca atc aaa 1680 Asp Gly Phe Tyr Ala Thr Ile ProGly Glu Lys Pro Glu Thr Ile Lys 545 550 555 560 aag aaa gct aag gaa ttctta aaa tac ata aac tcc aaa ctt ccc ggt 1728 Lys Lys Ala Lys Glu Phe LeuLys Tyr Ile Asn Ser Lys Leu Pro Gly 565 570 575 ctg ctc gag ctt gag tatgag ggc ttt tac ttg aga gga ttt ttc gtc 1776 Leu Leu Glu Leu Glu Tyr GluGly Phe Tyr Leu Arg Gly Phe Phe Val 580 585 590 gca aag aag cgc tat gcggtt ata gac gaa gaa ggt agg ata acg aca 1824 Ala Lys Lys Arg Tyr Ala ValIle Asp Glu Glu Gly Arg Ile Thr Thr 595 600 605 agg ggt ctg gaa gtt gtaagg agg gac tgg agc gaa ata gcc aaa gag 1872 Arg Gly Leu Glu Val Val ArgArg Asp Trp Ser Glu Ile Ala Lys Glu 610 615 620 acc cag gct aaa gtc ttggag gca ata ctt aaa gaa gat agt gtc gaa 1920 Thr Gln Ala Lys Val Leu GluAla Ile Leu Lys Glu Asp Ser Val Glu 625 630 635 640 aaa gct gtg gaa atcgtt aag gac gtt gtt gag gag ata gca aaa tac 1968 Lys Ala Val Glu Ile ValLys Asp Val Val Glu Glu Ile Ala Lys Tyr 645 650 655 caa gtc ccg ctt gaaaag ctt gtt atc cac gag cag att acc aag gat 2016 Gln Val Pro Leu Glu LysLeu Val Ile His Glu Gln Ile Thr Lys Asp 660 665 670 cta agt gaa tac aaagcc att ggg cct cat gta gca ata gca aag agg 2064 Leu Ser Glu Tyr Lys AlaIle Gly Pro His Val Ala Ile Ala Lys Arg 675 680 685 ctt gct gca aag ggaata aaa gtg aga ccc ggc acg ata ata agc tat 2112 Leu Ala Ala Lys Gly IleLys Val Arg Pro Gly Thr Ile Ile Ser Tyr 690 695 700 atc gtc ctc agg ggaagc gga aag ata agt gac agg gta att ttg ctt 2160 Ile Val Leu Arg Gly SerGly Lys Ile Ser Asp Arg Val Ile Leu Leu 705 710 715 720 tca gag tat gatccg aaa aaa cac aag tac gac ccc gac tac tac ata 2208 Ser Glu Tyr Asp ProLys Lys His Lys Tyr Asp Pro Asp Tyr Tyr Ile 725 730 735 gaa aac caa gttctg ccg gcg gtg ctt agg atc ctt gaa gcc ttc ggc 2256 Glu Asn Gln Val LeuPro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly 740 745 750 tac aga aaa gaggac tta aaa tac caa agc tca aaa cag gtt gga ctg 2304 Tyr Arg Lys Glu AspLeu Lys Tyr Gln Ser Ser Lys Gln Val Gly Leu 755 760 765 gac gcg tgg cttaag aag tag 2325 Asp Ala Trp Leu Lys Lys 770 34 774 PRT T. aggregans 34Met Ile Phe Asp Thr Asp Tyr Ile Thr Lys Asp Gly Lys Pro Ile Ile 1 5 1015 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Leu Asp Pro 20 2530 His Phe Gln Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 4045 Asp Glu Ile Lys Ala Ile Lys Gly Glu Arg His Gly Lys Ile Val Arg 50 5560 Val Val Asp Ala Val Lys Val Lys Lys Lys Phe Leu Gly Arg Asp Val 65 7075 80 Glu Val Trp Lys Leu Ile Phe Glu His Pro Gln Asp Val Pro Ala Leu 8590 95 Arg Gly Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Tyr Glu Tyr100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu IlePro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Leu Met Ala Phe Asp IleGlu Thr 130 135 140 Phe Tyr His Glu Gly Asp Glu Phe Gly Lys Gly Glu IleIle Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Glu Ala Arg Val IleThr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser AsnGlu Arg Glu Met Ile Lys 180 185 190 Arg Phe Val Gln Ile Val Arg Glu LysAsp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp LeuPro Tyr Leu Ile Lys Arg Ala Glu 210 215 220 Lys Leu Gly Val Thr Leu LeuLeu Gly Arg Asp Lys Glu His Pro Glu 225 230 235 240 Pro Lys Ile His ArgMet Gly Asp Ser Phe Ala Val Glu Ile Lys Gly 245 250 255 Arg Ile His PheAsp Leu Phe Pro Val Val Arg Arg Thr Ile Asn Leu 260 265 270 Pro Thr TyrThr Leu Glu Ala Val Tyr Glu Ala Val Leu Gly Lys Thr 275 280 285 Lys SerLys Leu Gly Ala Glu Glu Ile Ala Ala Ile Trp Glu Thr Glu 290 295 300 GluSer Met Lys Lys Leu Ala Gln Tyr Ser Met Glu Asp Ala Arg Ala 305 310 315320 Thr Tyr Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Glu Leu Ala 325330 335 Lys Leu Ile Gly Gln Ser Val Trp Asp Val Ser Arg Ser Ser Thr Gly340 345 350 Asn Leu Val Glu Trp Tyr Leu Leu Arg Val Ala Tyr Glu Arg AsnGlu 355 360 365 Leu Ala Pro Asn Lys Pro Asp Glu Glu Glu Tyr Arg Arg ArgLeu Arg 370 375 380 Thr Thr Tyr Leu Gly Gly Tyr Val Lys Glu Pro Glu ArgGly Leu Trp 385 390 395 400 Glu Asn Ile Thr Tyr Leu Asp Phe Arg Cys LeuTyr Pro Ser Ile Ile 405 410 415 Val Thr His Asn Val Ser Pro Asp Thr LeuGlu Arg Glu Gly Cys Lys 420 425 430 Asn Tyr Asp Val Ala Pro Ile Val GlyTyr Lys Phe Cys Lys Asp Phe 435 440 445 Pro Gly Phe Ile Pro Ser Ile LeuGly Glu Leu Ile Thr Met Arg Gln 450 455 460 Glu Ile Lys Lys Lys Met LysAla Thr Ile Asp Pro Ile Glu Lys Lys 465 470 475 480 Met Leu Asp Tyr ArgGln Arg Ala Val Lys Leu His Ala Asn Ser Tyr 485 490 495 Tyr Gly Tyr MetGly Tyr Pro Lys Ala Arg Trp Tyr Ser Lys Glu Cys 500 505 510 Ala Glu SerVal Thr Ala Trp Gly Arg His Tyr Ile Glu Met Thr Ile 515 520 525 Lys GluIle Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr 530 535 540 AspGly Phe Tyr Ala Thr Ile Pro Gly Glu Lys Pro Glu Thr Ile Lys 545 550 555560 Lys Lys Ala Lys Glu Phe Leu Lys Tyr Ile Asn Ser Lys Leu Pro Gly 565570 575 Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Leu Arg Gly Phe Phe Val580 585 590 Ala Lys Lys Arg Tyr Ala Val Ile Asp Glu Glu Gly Arg Ile ThrThr 595 600 605 Arg Gly Leu Glu Val Val Arg Arg Asp Trp Ser Glu Ile AlaLys Glu 610 615 620 Thr Gln Ala Lys Val Leu Glu Ala Ile Leu Lys Glu AspSer Val Glu 625 630 635 640 Lys Ala Val Glu Ile Val Lys Asp Val Val GluGlu Ile Ala Lys Tyr 645 650 655 Gln Val Pro Leu Glu Lys Leu Val Ile HisGlu Gln Ile Thr Lys Asp 660 665 670 Leu Ser Glu Tyr Lys Ala Ile Gly ProHis Val Ala Ile Ala Lys Arg 675 680 685 Leu Ala Ala Lys Gly Ile Lys ValArg Pro Gly Thr Ile Ile Ser Tyr 690 695 700 Ile Val Leu Arg Gly Ser GlyLys Ile Ser Asp Arg Val Ile Leu Leu 705 710 715 720 Ser Glu Tyr Asp ProLys Lys His Lys Tyr Asp Pro Asp Tyr Tyr Ile 725 730 735 Glu Asn Gln ValLeu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly 740 745 750 Tyr Arg LysGlu Asp Leu Lys Tyr Gln Ser Ser Lys Gln Val Gly Leu 755 760 765 Asp AlaTrp Leu Lys Lys 770

What is claimed is:
 1. A thermostable mutant B-type DNA polymerasecomprising a Y-GG/A amino acid motif between the N-terminal3′-5′-exonuclease domain and the C-terminal polymerase domain whereinthe tyrosine of the motif is substituted with another amino acid.
 2. Themutant B-type DNA polymerase according to claim 1 wherein the tyrosineof the motif is substituted with an amino acid with an aromatic sidechain.
 3. The mutant thermostable B-type DNA polymerase according toclaim 1 having a Y→F,Y→W or Y→H mutation.
 4. The mutant B-type DNApolymerase according to claim 1 wherein the tyrosine of the motif issubstituted with an amino acid with an hydrophilic side chain.
 5. Themutant thermostable B-type DNA polymerase according to claim 1 having aY→N or Y→S mutation.
 6. The mutant thermostable B-type DNA polymeraseaccording to claim 1 wherein its wild type form is obtainable fromEuryarchaea.
 7. The mutant thermostable B-type DNA polymerase accordingto claim 1 wherein its wild type form is obtainable from Thermococcusaggregans.
 8. The mutant of a thermostable B-type DNA polymeraseaccording to claim 1 wherein the amino acid sequence of its wild typeform is ≧80% homologous to the amino acid sequence of wild type Tag DNApolymerase.
 9. A DNA encoding a thermostable mutant DNA polymerase ofclaim
 1. 10. A vector containing the DNA according to claim
 9. 11. Atransformed host cell comprising the vector according to claim
 10. 12. Aprocess for obtaining a polymerase according to claim 1 comprising thesteps of cloning and mutagenesis of the gene, followed by the expressionand purification of the protein.
 13. A method of using the polymeraseaccording to claim 1 for synthesizing nucleic acids.
 14. A method ofusing the polymerase according to claim 1 for polymerase chainreactions.